Journal: Biotechniques

Article Title: Improved SARS-CoV-2 PCR detection and genotyping with double-bubble primers

doi: 10.2144/btn-2021-0063

Figure Lengend Snippet: (A–C) Dose–response curves using double-bubble primer mix #8 and VIC-TqM probe #12 and decimal dilutions of SARS-CoV-2 synthetic RNA N. (A) Real-time amplification plot. C t values: 8 × 10 5 copies, 22.82; 8 × 10 4 copies, 26.27; 8 × 10 3 copies, 30.55; 8 × 10 2 copies, 32.51. (B) 5% agarose gel of the PCR amplification products. Lane 1: ultra-low-range ladder; lanes 2–5: 139-bp amplicons of 8 × 10 5 to 8 × 10 2 RNA copies per tube. Also included in the PCR reaction mix were 2× TqM buffer without UDP and iScript reverse-transcriptase. (C) Efficiency plot of the PCR reaction depicted in panel A. Slope = -3.3359 represents PCR reaction efficiency of 99.42%; R 2 = 0.9816. (D) One-tube RT-qPCR using double-bubble primer mix #10 from region 2 using SARS-CoV-2 synthetic RNA (2 × 10 4 copies per tube) and FAM-TqM probe #13. RT-qPCR was performed with standard conditions.

Article Snippet: For cDNA synthesis, iScript reverse-transcriptase kit was employed: 25°C, 5 min, 42°C, 15 min 95°C, 1 min, 4°C hold (BioRad, 1708891).

Techniques: Amplification, Agarose Gel Electrophoresis, Reverse Transcription, Quantitative RT-PCR

Journal: Biotechniques

Article Title: Improved SARS-CoV-2 PCR detection and genotyping with double-bubble primers

doi: 10.2144/btn-2021-0063

Figure Lengend Snippet: (A) qPCR using reverse-transcribed cDNA template and double-bubble (D-B) primer mix #10, FAM-TqM probe #13, TqM fast kit+ uracil-DNA glycosylase (UDG) assayed in duplicates. Insert depicts 5% agarose gel; lane 1: ultra-low-range (ULR) ladder; lanes 2 & 3: template cDNA; lanes 4 & 5: non-template control (NTC). C t average values: cDNA, 28.60; NTC, undetermined. (B) One-tube RT-qPCR using SARS-CoV-2 synthetic RNA N template and D-B primer mix #8, VIC-TqM probe #12, TqM fast kit without UDG iScript reverse transcriptase, assayed in duplicate. Insert depicts 5% agarose gel; lane 1: ULR ladder; lanes 2 and 3: template RNA; lanes 4 and 5: NTC. C t average values: RNA, 24.63, NTC, undetermined. (C) Duplex qPCR using SARS-CoV-2 synthetic RNA N template and TqM fast kit with UDG. C t values: primer 8, 26.13; primer 10, 32.19; duplex primers 8 + 10, 26.36 and 30.27, respectively; NTC, undetermined. (D) 5% agarose gel of samples shown in panel C. Lane 1: ULR ladder; lane 2: primer D-B mix #8 (amplicon 139 bp); lane 3: D-B primer mix #10 (amplicon 158 bp); lane 4: duplex of both primers #8 and #10 (amplicons 139 and 158 bp). Arrows (C & D) show duplex amplifications in the same tube with D-B primers #8 and #10. PCR was performed using fast conditions with 40 (A, C & D) and 30 (B) cycles.

Article Snippet: For cDNA synthesis, iScript reverse-transcriptase kit was employed: 25°C, 5 min, 42°C, 15 min 95°C, 1 min, 4°C hold (BioRad, 1708891).

Techniques: Reverse Transcription, Agarose Gel Electrophoresis, Control, Quantitative RT-PCR, Amplification

Journal: Biotechniques

Article Title: Improved SARS-CoV-2 PCR detection and genotyping with double-bubble primers

doi: 10.2144/btn-2021-0063

Figure Lengend Snippet: The reaction mixture contained hot-start double-bubble (D-B) primer mix #4, VIC-TqM probe #12, cost-effective non-hot-start Taq polymerase (FroggaBio), reverse transcriptase (iScript), synthetic SARS-CoV-2 RNA gene N as template, added dNTPs and ROX dye for internal calibration. The reaction was assembled at room temperature and subjected to real time RT-qPCR using the following fast conditions: 42°C, 15 min; 95°C,1 min; 30 cycles of 95°C, 1 s → 70°C, 20 s. The amplification plot and (insert) 5% agarose gel depict the amplification of SARS-CoV-2 RNA extracted from the nasopharyngeal swabs of patient S1 (lane 2) and negative patient N1 (lane 3), no-template controls (lanes 4 and 5) and SARS-CoV-2 gene N synthetic RNA as positive control (lane 6). Note the amplification in the real-time plot and the 115-bp band in the 5% agarose gel with SARS-CoV-2 virus or synthetic RNA (lanes 2 and 6), with no amplifications in the negative control N1 (lane 3) or NTC (lanes 4 and 5). Lane 1: ultra-low-range ladder. C t values: positive patient S1: 23.95; synthetic RNA: 23.84; negative patient N1 and NTC: undetermined.

Article Snippet: For cDNA synthesis, iScript reverse-transcriptase kit was employed: 25°C, 5 min, 42°C, 15 min 95°C, 1 min, 4°C hold (BioRad, 1708891).

Techniques: Reverse Transcription, Quantitative RT-PCR, Amplification, Agarose Gel Electrophoresis, Positive Control, Virus, Negative Control